The Fate of a Hapten

www.nature.com/scientificreports

OPEN The Fate of a Hapten - From the Skin to Modifcation of Macrophage Migration Inhibitory Factor (MIF) in Received: 14 September 2017 Accepted: 31 January 2018 Lymph Nodes Published: xx xx xxxx Isabella Karlsson 1, Kristin Samuelsson2, Carl Simonsson2, Anna-Lena Stenfeldt2, Ulrika Nilsson1, Leopold L. Ilag1, Charlotte Jonsson2 & Ann-Therese Karlberg2

Skin (contact) allergy, the most prevalent form of immunotoxicity in humans, is caused by low molecular weight chemicals (haptens) that penetrate stratum corneum and modify endogenous proteins. The fate of haptens after cutaneous absorption, especially what protein(s) they react with, is largely unknown. In this study the fuorescent hapten tetramethylrhodamine isothiocyanate (TRITC) was used to identify hapten-protein conjugates in the local lymph nodes after topical application, as they play a key role in activation of the adaptive immune system. TRITC interacted with dendritic cells but also with T and B cells in the lymph nodes as shown by fow cytometry. Identifcation of the most abundant TRITC-modifed protein in lymph nodes by tandem mass spectrometry revealed TRITC-modifcation of the N-terminal proline of macrophage migration inhibitory factor (MIF) – an evolutionary well-conserved protein involved in cell-mediated immunity and infammation. This is the frst time a hapten-modifed protein has been identifed in lymph nodes after topical administration of the hapten. Most haptens are electrophiles and can therefore modify the N-terminal proline of MIF, which has an unusually reactive amino group under physiological conditions; thus, modifcation of MIF by haptens may have an immunomodulating role in contact allergy as well as in other immunotoxicity reactions.

About 20% of the population in the Western world have skin (contact) allergy to one or more compounds in their close environment1. Allergic contact dermatitis (ACD), i.e. skin infammation and eczema, is the clinical manifestation of contact allergy and afects 5–10% of the population2,3. ACD is caused by T-lymphocyte mediated type IV hypersensitivity responses to antigens afer skin exposure to contact allergens. Small (<1000 Da) reactive organic molecules (haptens) able to penetrate stratum corneum (SC), the top-most layer of the skin and covalently modify endogenous proteins are the most important contact allergens from a clinical point of view. Hapten exposure leads to activation of cutaneous dendritic cells (DCs), which migrate from the skin to the draining lymph nodes (LNs) where they present peptides (potential antigens) from hapten-modifed proteins to naïve T cells; thereby, activating the adaptive (acquired) immune system. Te naïve T cells that have recognized an antigen start to proliferate and diferentiate into antigen-specifc efector and memory T cells that circulate in the blood and lymphatic system3–5. Many aspects of the mechanisms underlying ACD are still unknown despite recent advances in understanding the role and interaction of diferent immune cells. For instance, although the hypothesis that haptens induce immune responses by modifying endogenous proteins was introduced by Landsteiner and Jacobs already in 19366 the identity of these modifed proteins has remained more or less unknown even until today. In particular, knowledge of the identity of hapten-protein conjugates seen as immunogenic by the immune system, i.e. that become antigens that activate naïve T cells, will increase our mechanistic understanding of contact allergy. In addition these hapten-protein conjugates could have potential as biomarkers for the development of better diagnostic tests for contact allergy and may also prove useful in the development of improved treatments. One previously unexplored approach to fnd potentially immunogenic hapten-modifed proteins would be to identify hapten-protein conjugates in the LNs afer topical administration.

1Department of Environmental Science and Analytical Chemistry, Stockholm University, Stockholm, Sweden. 2Department of Chemistry and Molecular Biology, Dermatochemistry, University of Gothenburg, Gothenburg, Sweden. Correspondence and requests for materials should be addressed to I.K. (email: [email protected])

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 1 www.nature.com/scientificreports/

Te aim of the present study was to improve our understanding regarding the fate of a specifc hapten afer topical application and, in particular, to identify hapten-protein conjugates in LNs as these play a key role in activation of the adaptive immune system. Te hapten-protein conjugates (adducts) are most ofen formed by reactions between electrophilic haptens and nucleophilic side chains such as cysteines (thiols) and lysines (primary amines) in skin proteins. Isothiocyanate (NCS) is an electrophilic functional group prone to react with both amines and thiols. Fluorescent isothiocyanates, e.g. fuorescein isothiocyanate (FITC), are due to their reactivity with proteins extensively used in various biological applications. In the present study mice were topically exposed to two fuorescent compounds, tetramethylrhodamine isothiocyanate (TRITC, sensitizer)7 and tetraethylrhodamine (Rhodamine B, non-sensitizer) and the distribution in skin and draining LNs was investigated. Microscopic examination of skin penetration of TRITC afer topical application showed that the most intense fuorescence signal was detected in SC. Excision and inspection of the draining LNs from mice sensitized to TRITC revealed fuorescence in DCs but also in T and B cell populations. No fuorescence was detected in LNs in mice treated with Rhodamine B. Detection of TRITC-modifed proteins isolated from LN cells, followed by identifcation of the most strongly TRITC-fuorescent protein, revealed TRITC bound to the N-terminal proline of the pleiotropic cytokine macrophage migration inhibitory factor (MIF). To the best of our knowledge, this is the frst time a hapten-protein conjugate has been identifed in local LNs afer topical application of the hapten. Results In the current project, the fuorescent skin sensitizer TRITC (Fig. 1) and the structurally similar compound Rhodamine B (Fig. 1), which is fuorescent but lacks the reactive NCS group of TRITC, were topically applied to the dorsum of mice ears for three consecutive days. Tereafer, the TRITC/Rhodamine B fuorescence in skin, draining LNs, LN cells and LN proteins was investigated.

Sensitization assessment. Te skin sensitizing potential of Rhodamine B was investigated in the Local Lymph Node Assay (LLNA)8–10, which is the recommended OECD method for assessing the skin sensitizing potential of a compound. Rhodamine B was considered a non-sensitizer in the used test concentrations (up to 5% (w/v)) as no cell proliferation was induced in the LNs (Supporting Information, Table S1). Tis is in contrast to TRITC, which we have previously shown to be an extreme skin sensitizer in the LLNA with an EC3 value of 0.040%7.

Visualization of fuorophore distribution in mouse ears. To study the distribution of TRITC and Rhodamine B in the skin afer topical in vivo exposure, sections of fuorophore-exposed mouse ears were investigated using laser scanning confocal microscopy (LSCM). TRITC was detected mainly in SC, in hair follicles, in the matrix of the cartilage and to a minor extent in viable epidermis (Fig. 1a). To visualize the fuorescence from the control substance Rhodamine B, the laser power had to be increased 20 times compared to the settings for the TRITC sample. Te most intense Rhodamine B fuorescence was seen in hair follicles, while fuorescence in SC and cartilage was hardly detectable despite the increased laser power (Fig. 1b). Te discrepancy between the distributions of the two fuorophores in the skin is likely due to the NCS group’s ability to covalently modify biomacromolecules. We have previously shown in in vitro studies on human skin using LSCM and two photon microscopy that FITC, structurally similar to TRITC with corresponding skin sensitizing properties11, accumu- lated in SC while fuorescein, the non-sensitizing analogue lacking the NCS group, penetrated to deeper layers of epidermis12.

Visualization of fuorophores in LNs and distribution in LN cells. To further investigate the distribution of the fuorophores in mice, ear draining LNs were excised and analyzed with LSCM and fow cytometry. Te presence of TRITC was clearly visualized in LN sections (Fig. 1c), whereas the negative control Rhodamine B was not observed using LSCM (data not shown). Te number of LN cells was markedly increased in mice treated with TRITC and the proportion of fuorescent LN cells was estimated to on average 77% by fow cytometry (Table S2). Te number and fuorescence of LN cells in Rhodamine-exposed mice was the same as for vehicle-treated mice; hence corroborating the LSCM study in which no Rhodamine B fuorescence was detected in the LN section. Earlier skin painting studies with TRITC have identifed DCs as responsible for the accumulation of the fluorophore in LNs13–16. Indeed, our study shows that almost all DCs were TRITC positive (around 85%). However, our investigation analyzed diferent cell types and could show that T and B cells were the major cellular targets of the fuorophore (Fig. 2). While DCs constituted a minor part (2–3%) of the LN cell population, T-cells were the main cell population (around 90%) in the LNs of the TRITC exposed mice and two thirds of the T-cells were TRITC positive. B cells constituted about 10% of the LN cells and of these more than half were TRITC positive. Tus, the high yield of TRITC positive cells in the LN is unlikely to solely depend on transport of haptenated proteins/peptides from the skin by LN immigrating DCs. Studies of other fuorescent isothiocyanates, by us17 and others18,19, have shown that in addition to the relatively slow cell-mediated transport of these compounds to the local LNs by DCs, they are also transported as free haptens or soluble hapten-protein complexes via the lymph and blood to the secondary lymphoid organs. In the LNs, these soluble haptens/hapten-protein conjugates might be captured by antigen-presenting cells such as resident DCs, macrophages, or B cells.

Hapten-modified proteins in LNs. To identify which proteins in the LN cells that were modified by TRITC, the following experiments were conducted. Lysed single-cell suspensions from LNs of TRITC- or vehicle-treated mice were separated using gel electrophoresis. Fluorescent proteins were visualized by scanning at 532 nm (excitation)/580 nm (emission) (Fig. 3). One strong fuorescent protein band and several weaker fuorescent protein bands were visualized in the gel separation of LN proteins from TRITC exposed mice (Fig. 3, panel 1 – TRITC fuorescence, well 2). No fuorescent bands could be detected in the gel separation of LN proteins from the vehicle-exposed mice (Fig. 3, panel 1 – TRITC fuorescence, well 3).

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 2 www.nature.com/scientificreports/

Figure 1. Laser scanning confocal microscopy (LSCM) images of cryosectioned tissues. Mice were exposed to 25 µl of tetramethylrhodamine isothiocyanate (TRITC) (11 mM) (a,c) or the non-sensitizing structural analogue tetraethylrhodamine (Rhodamine B) (11 mM) (b) on the dorsal side of each ear for three consecutive days. Eighteen hours afer the last exposure, the mice were sacrifced and ears (a,b) and draining lymph nodes (c) were excised, snap frozen and sectioned (10 µm and 14 µm, respectively). Slides were fxed in acetone. To be able to detect Rhodamine B, the laser intensity was increased 20 times compared to the settings for TRITC. Te scale bars refer to 75 µm (a,b) and 500 µm (c). h, hair follicle; ca, cartilage; e, epidermis; sc, stratum corneum. Te structures of TRITC and Rhodamine B are shown at the top of the fgure.

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 3 www.nature.com/scientificreports/

Figure 2. Flow cytometry analysis of lymph node cell populations. Cells from ear draining lymph nodes isolated from mice exposed to TRITC (5.6 mM in acetone:dibutyl phthalate, 1:1, 25 µl) (a–d) or vehicle (acetone:dibutyl phthalate, 1:1, 25 µl) (e–h) on both ears for three consecutive days were analyzed by fow cytometry. Lymph node cells from each treatment group were pooled before analysis. Te dot plots show unstained cells (a,e) and cells stained with allophycocyanin (APC) labeled antibodies toward T cells (CD3ε; b,f), B cells (CD19; c,g), or DC (CD11c; d,h). Q1 (upper lef quadrant) shows the proportion of APC positive/TRITC negative cells and Q2 (upper right) shows the proportion of double positive cells. Q3 (lower lef) shows the proportion of double negative cells and Q4 (lower right) the proportion of APC negative/TRITC positive cells.

Te most intense fuorescent band (10–15 kDa) was excised, digested and analyzed with nanofow liquid chromatography (nanoLC) and hybrid linear ion-trap Fourier-transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). With the use of the sofware MASCOT the acquired tandem mass spectra of the tryptic peptides were searched against the Swiss prot database, which resulted in the identifcation of 10 diferent proteins (Table 1). Tis procedure only identifed known tryptic peptides and matched them with the protein database, i.e. although modifed peptides could not be identifed with this method, it is still possible that one or several of these proteins are TRITC-modifed. Of the identifed proteins, MIF was of particular interest for two main reasons, 1) MIF is known to play an important roll in infammatory and immune diseases20–24 and 2) we have previously shown that TRITC is very reactive toward N-terminal prolines and that the formed conjugates (TRITC-thiourea) are stable adducts7. MIF has an N-terminal proline with a remarkably low pKa of 5.625, which turns the amino group into an unusually strong nucleophile under physiological conditions. Interestingly, reports in the literature describe specifc modifcation of MIF by other compounds with an isothiocyanate functional- ity26–28. Western blot immunostaining with a MIF polyconal antibody was used to verify the co-localization of MIF with the most intense TRITC fuorescent band (Figure S1). To confrm that TRITC is actually bound to MIF, the suspected TRITC-modifed MIF peptide (TRITC-PMFIVNTNVPR) was generated and used as a reference compound in a targeted screening for TRITC-MIF conjugates in the LN fractions using ultra performance liquid chromatography (UPLC) and electrospray (ESI) quadrupole time-of-fight (qToF) MS/MS. Te analyses showed that TRITC bound to the N-terminal proline of MIF was present in the B fraction (Fig. 3) of the 10–15 kDa section of the LN proteins from TRITC-treated animals. Te developed MS/MS method uses a fxed mass (in the frst quadrupole) of 865.9 for TRITC-MIF and 644.3 for non-modifed MIF; both masses correspond to doubly charged molecular ions. Te displayed chromatograms in Fig. 4a (reference compound) and Fig. 4b (B fraction)

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 4 www.nature.com/scientificreports/

Figure 3. Gel separation of proteins from lymph node cells. Lymp node cell proteins were isolated from pooled lymph node cells from mice exposed to vehicle (acetone:dibutyl phthalate, 1:1) or TRITC (5.6 mM) on the dorsum of the ears for three consecutive days. Proteins were separated using 1D SDS-PAGE and TRITC fuorescence (panel 1) was visualized using a fuorescence scanner with a GreenLED 528 nm laser with a bandpass flter of 605 ± 50 nm. Each well was loaded with 100 µg of lymph node protein from TRITC (well 2) or vehicle (well 3) exposed mice. Te Novex Sharp pre-stained standard was used as a molecular standard (well 1). Te total protein contents in wells 2 and 3 were visualized with SimplyBlue staining (panel 2).

Uniprot number Protein name MW (kDa) PI(calculated) Score No of peptides P62962 Proflin-1 14.9 8.28 674.16 4 P02088 Hemoglobin subunit beta-1 15.8 7.65 366.52 6 Macrophage migration inhibitory P34884 12.5 7.34 230.42 2 factor O35215 D-Dopachrome decarboxylase 13.1 6.54 188.14 5 Q9ERR7 15 kDa Selenoprotein 17.8 5.35 183.54 3 Heterogeneous nuclear P61979 50.9 5.54 178.32 2 ribonucleoprotein K P02089 Hemoglobin subunit beta-2 15.9 8.05 128.04 3 P61971 Nuclear transport factor 2 14.5 5.38 109.47 2 P62806 Histone H4 11.4 11.36 60.87 2 P62270 40 S Ribosomal protein S18 17.7 10.99 42.05 2

Table 1. Identifcation of potential TRITC-labeled lymph node cell proteins using nanoLC-MS/MS.

represent extracted masses corresponding to the TRITC-fragments (m/z = 444.14, singly charged ion), the MIF peptide-fragment (m/z = 1287.68, singly charged ion), as well as the full TRITC-MIF conjugate (m/z = 865.91, doubly charged ion). In Fig. 4c,d, the spectra from the reference compound and the corresponding peaks in fraction B are shown. Two peaks with diferent retention times, but the same exact mass and fragmentation pattern were seen. In our study the mice were treated with commercially obtained TRITC consisting of a mixture of six isomers. Te reference TRITC-peptide (TRITC-PMFIVNTNVPR), which was prepared from the same mix of TRITC isomers, gave the same two peaks as those observed in the B fraction of the 10–15 kDa sample (Fig. 4). No TRITC-modifed MIF peptide (TRITC-PMFIVNTNVPR) was detected in the other two fractions (A and C) of the 10–15 kDa sample. Non-modifed MIF peptide (PMFIVNTNVPR) was detected in fraction A only (Figure S2). With the use of a standard curve (Figure S3), the concentration of PMFIVNTNVPR in fraction A was determined to be around 7–8 nM (see Supplementary Information). By comparison with the reference sample and the assumption that all depletion of PMFIVNTNVPR in the reference sample was due to reaction with TRITC, the concentration of TRITC-PMFIVNTNVPR in fraction B was estimated to be around 2–3 nM (see Supplementary Information). Tus, the portion of modifed MIF in the LN would be around 20–30% of the total amount of MIF (non-modifed MIF (7–8 nM) plus TRITC-modifed MIF (2–3 nM)). Discussion Although contact allergy is the most prevalent form of immunotoxicity in humans and involves about a ffh of the population in the Western world there is a remarkable lack of mechanistic knowledge. A recent review by Koppes et al. concludes that despite the vast amount of research into skin sensitization, promising specifc biomarkers

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 5 www.nature.com/scientificreports/

Figure 4. Chromatogram and spectra of TRITC-modifed MIF chromatograms and spectra of the reference compound (TRITC bound to the N-terminal MIF peptide PMFIVNTNVPR) and fraction B from lymph node cell proteins isolated from TRITC-exposed mice and separated with 1D SDS-PAGE. Te selected bands (see Fig. 3) were excised, digested and analyzed with UPLC-qToF. A reference compound was prepared to confrm the identity of the peptide. Te qToF was operated in positive resolution mode with electrospray ionization. A qToF ms/ms method was used where the fxed mass was set to 865.9, the ms2 was set to 50–1500 m/z and the collision energy ramp was from 15 eV to 40 eV. Te extracted chromatograms (a,b) correspond to the TRITC- fragment [m/z = 444.14, singly charged ion], the MIF peptide-fragment [m/z = 1287.68, singly charged ion] and the full TRITC-MIF conjugate [m/z = 865.91, doubly charged ion]. (a) Chromatogram of reference compound, (b) chromatogram of fraction B, (c) spectrum of reference compound and (d) spectrum of fraction B.

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 6 www.nature.com/scientificreports/

for ACD have not yet been described29. In particular, there is a dearth of knowledge concerning the identity of hapten-protein complexes that are immunogenic. Reactive organic chemicals can modify proteins in the skin – a feature which is crucial for the activation of the innate immune system3–5. However, these hapten-protein conjugates may not be identical to the antigens that activate the naïve T cells in the LNs i.e. hapten-modifed proteins involved in activating the adaptive immune response. Whether the hapten-protein conjugates identifed in earlier research focusing on identifying protein targets in skin30 and in vitro31–35, are actually involved in activation of the adaptive immune system is unknown. In the current study, the objective was to identify hapten-protein conjugates in LNs rather than in the skin with the rationale that a hapten-modifed protein in the LNs may play a key role in activation of the adaptive immune system and may therefore be a biomarker for ACD. Te homotrimeric protein MIF is known to have keto-enol tautomerase activity, which is catalyzed by its N-terminal proline36–38. Analysis of the three-dimensional structure of MIF has shown that a solvent accessible pocket is formed around the N-terminal proline forming the catalytic site. Te hydrophobic residues around the N-terminal proline in the active site lower the pKa of the amino group to such an extent that it is unprotonated at physiological pH25. Tis makes the N-terminal proline of MIF signifcantly more reactive than lysine amino groups, which normally are protonated under physiological conditions. Traditionally, experiments to identify targets for haptens have mainly focused on reactivity toward cysteine and lysine and although most haptens have been shown to be more reactive toward cysteine than lysine, we as well as others have demonstrated that hapten-cysteine adducts are ofen unstable7,39–41. Tere are, however, some studies by us and others in which the reactivity of the peptide PHCKRM toward a number of haptens, such as isothiocyanates7, epoxides42–44, oximes45, and anhydrides41 has been investigated. Tese studies found that the investigated haptens formed stable adducts by covalent binding to the N-terminal proline of PHCKRM. Te results from the earlier investigations are in line with the present fndings; although, there are diferences, especially in 3D structure, between the N-terminal proline of the linear synthetic peptide PHCKRM and the N-terminal proline of MIF situated in a catalytic pocket. MIF is expressed by more or less all immune cells such as T cells, B cells, DCs, monocytes, macrophages, neu- trophils, eosinophils, basophil and mast cells; in addition, MIF is expressed in tissues that come in direct contact with our external environment, such as the skin22. Tus, it is possible that MIF could be a target protein for most contact allergens, either via a direct reaction with the N-terminal proline or via a primary, unstable, cysteine binding. Further support for this hypothesis is that contact hypersensitivity toward the haptens trinitrochlorobenzene and oxazolone has been shown to be impaired in MIF defcient mice46. Interestingly, it has been suggested that MIF requires a co-factor, such as a substrate for a chemical reaction, in order to induce immune reactions25. Hence, one could hypothesize that haptens, by covalent binding to the N-terminal proline in the catalytic site of MIF, might function as such co-factors. Another hypothesis is that TRITC or TRITC-MIF, in addition to the classic major histocompatibility complex (MHC) – T cell receptor (TCR) pathway, is able to activate T cells via the so-called p-i mechanism (pharmacological interaction with immune receptors)47. Te p-i mechanism, which states that certain compounds can bind directly to T cell receptors (TCRs) and MHC molecules, has been suggested for T cell mediated drug hypersensitivity as well as ACD48–51. Te p-i concept bears strong similarities to super-antigen stimulations52, which are caused by antigens produced by certain pathogenic viruses and bacteria. Support for the hypothesis that TRITC or TRITC-MIF can activate T cells via the p-i mechanism can be found in the literature. In a study by Diamond et al., FITC-conjugates were shown to activate FITC-specifc T cells through direct binding to the TCRs without involvement of MHC53. Importantly though, the p-i concept also states that direct binding of drugs/haptens/ haptenated proteins to naïve T cells is, in general, not enough to initiate an immune response. However, binding to primed T cells, which have a lower threshold than naïve T cells, could lead to further T cell stimulation and expansion47,54. Tus, it would appear as if the p-i efect is most important in the efector phase of ACD when memory T cells are already present. Nevertheless, TRITC-MIF binding to T cells would still occur in the sensitization phase. As T cells can bind MIF via CXCR455, the large population of TRITC-labeled T- and B-cells detected in the LNs (Fig. 2) might be due to TRITC-MIF interaction with Ii, CXCR4, or any other MIF receptors that lymphocytes express. To the best of our knowledge, this is the frst time that a hapten-modifed protein has been identifed in local LNs afer topical application. Tus, it is possible that hapten-modifed MIF is a new biomarker for ACD; however, studies of other haptens would need to be conduct to investigate if hapten-modifcation of MIF is a general feature in contact allergy. MIF secretion has been correlated to a number of autoimmune and infammatory diseases, including atopic dermatitis24, arthritis56, colitis57, sepsis58,59, and even various cancers and their control by the immune system60–64. Tus, MIF’s role as a hapten target, its potential involvement in the formation of immunogenic complexes and its immunomodulatory role in the innate and adaptive immune systems may be of signifcance not only in ACD but also in many other diseases. Hence, this work should provide a basis for investigation of several aspects of hapten-modifcation of MIF in immunotoxicity, such as: a) the role of proline in formation of immunogenic complexes, b) how administration of hapten-modifed MIF, instead of hapten, would afect the potency of contact sensitization, c) where and when the MIF-hapten conjugates are formed, d) the role of diferent types of hapten bindings (e.g. covalent binding to proteins vs. p-i efects) and e) immunoregulation by haptenated proteins in immunotoxicity beyond contact sensitization. Material and Methods Chemicals. Acetone was purchased from Merk (Darmstadt, Germany). Tetramethylrhodamine isothiocyanate (TRITC), tetraethylrhodamine (Rhodamine B), dibutyl phthalate, phosphate-bufered saline (PBS) tab- lets (without Mg2+ and Ca2+), trichloroacetic acid (TCA) (>99%), dithiothreitol (DTT) (>99%), ammonium bicarbonate (99%), iodoacetamide (IAA) (>99%) and Clarion mounting medium, were purchased from Sigma- Aldrich Chemie (Steinheim, Germany). [Methyl-3H] thymidine was purchased from Amersham Biosciences (Buckinghamshire, UK). Peptide PMFIVNTNVPR was obtained from Peptide 2.0 (Chantilly, VA). Trypsin was

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 7 www.nature.com/scientificreports/

purchased from Roche Applies Science (Mannheim, Germany). Trypsin/Lys-C mix was obtained from Promega (Madison, WI).

Animals. Female CBA/Ca mice (B&K Scanbur, Sollentuna, Sweden) 8–12 weeks of age were used. The mice were housed in cages with HEPA-fltered airfow under conventional conditions in light-, humidity- and temperature-controlled rooms. Te regional ethics committee, Jordbruksverket, approved all experimental pro- tocols and the animal procedures were carried out in accordance with the approved guidelines.

LLNA. Te sensitizing potential of Rhodamine B was determined using the LLNA8–10 (see Supplementary Materials online for details).

Treatment of mice and tissue collection for fuorophore distribution studies. Groups of mice were topically exposed to 25 µl of TRITC (0.25% (w/v), 5.6 mM, 12 mice or 0.50%, 11 mM, 6 mice), Rhodamine B (0.54%, 11 mM, 6 mice), or vehicle (acetone:dibutyl phtalate, 1:1, 18 mice), on the dorsum of the ears for three consecutive days. Eighteen hours afer the last exposure, the mice were sacrifced and the ears and the auricular LNs were excised (see Supplementary Materials online for details).

LSCM of ear- and LN sections. Frozen ears and LNs were sectioned into 10 µm and 14 µm thick samples, respectively, on a cryostat (Leica Microsystems, Wetzlar, Germany). Imaging of ear and LN thin sections was performed using an LSCM 510 Meta system (Zeiss, Jena, Germany), at the Centre for Cellular Imaging, University of Gothenburg, Sweden (see Supplementary Materials online for details).

Detection of fuorescent draining LN cells using fow cytometry. Single cell suspensions of LN cells were washed with PBS and diluted to 106 cells ml−1 in PBS. Te proportion of fuorescent LN cells from mice exposed to TRITC (11 mM) was compared to the proportion in mice exposed to Rhodamine B (11 mM). Te distribution of TRITC in diferent LN cell populations from mice exposed to TRITC (5.6 mM) was investigated using typical markers for B-cells (CD19), T-cells (CD3ε) and dendritic cells (CD11c). Cells from vehicle-treated mice were used as a negative control. Te analyses were conducted with a FACSArray fow cytometer (BD Biosciences) equipped with a 532 nm excitation laser (see Supplementary Materials online for details).

Detection of TRITC-modifed proteins in LN cells. LN single cell suspensions from TRITC- or vehicle- exposed mice were pooled group-vise and lysed using Pierce IP Lysis bufer (Fischer scientifc) supplied with Halt Protease Inhibitor Cocktail (Fischer Scientifc), according to the manufacturer’s instructions and stored at −80 °C until used for protein analyses. Protein contents were estimated using Pierce BCA protein assay reagent kit with BSA as standard (Fischer Scientifc) according to the manufacturer’s instructions. In accordance with Invitrogen NuPAGE gel electrophoresis guide, proteins (100 μl/well) were separated on a 1 mm 4–12% gradient Bis-Tris gel (Invitrogen NuPAGE) separating 2–200 kDa proteins. Proteins were dissolved in Invitrogen NuPAGE LDS sample bufer containing DTT (50 mM) and each well was loaded with 100 µg protein. Te gel was run in dark- ness for 37 min at 200 V in a Novex mini cell electrophoresis system (Invitrogen) using Novex Sharp pre-stained standard (Invitrogen) as reference. Fluorescent proteins were detected using a fuorescence scanner, Versa Doc 4000 (Bio-Rad) equipped with a GreenLED, 528 nm laser and a bandpass flter (605 ± 50 nm) and analyzed with the sofware Quantity One 4.6.9. To confrm an even protein loading in the wells, the gel was stained using SimplyBlue SafeStain (Invitrogen).

Protein identifcation using nanoLC/ESI-MS/MS. Te gel piece between 10–15 kDa (containing the most intense TRITC-fuorescent band) was excised and in-gel digestion, including reduction with DTT and alkylation with IAA, was performed using Trypsin (Roche Applies Science) according to the manufacturer’s instructions. Extracted peptides were lypholized using a DNA120 SpeedVac (Termo Scientifc) and reconstituted in 0.1% formic acid (aq.). Te sample was analyzed on a nanoLC system coupled to a hybrid linear ion-trap FT-ICR mass spectrometer equipped with a 7 T magnet (LTQ-FT, Termo Scientifc). Te nanoLC system was equipped with an HTC-autosampler connected to an Agilent 1100 binary pump on which the peptides were trapped on a precolumn (45 × 0.075 mm i.d.) and separated on a reversed phase column 200 × 0.050 mm, both packed in-house with 3 µm Reprosil-Pur C18-AQ particles. A 40 min gradient from 10% to 50% acetonitrile in water containing 0.2% formic acid was used for separation of peptides. Te fow through the analytical column was 100 nl/ min and the injection volume was 2 µl. Te mass spectrometer was operated in data-dependent mode, automat- ically switching to MS/MS mode. MS-spectra were acquired in the LTQ-trap. For each scan of FT-ICR, the three most intense doubly or triply charged ions were sequentially fragmented in the linear trap by collision-induced dissociation. All the tandem mass spectra were searched by MASCOT (Matrix Science) against the Swiss prot database 57.1. Te search parameters were set to: All species, MS accuracy 5 ppm, MS/MS accuracy 0.5 Da, one missed cleavage by trypsin allowed, fxed modifcation of propionamide modifcation of cysteine and variable modifcation of oxidized methionine. For protein identifcation the minimum criteria were two tryptic peptides matched at or above 95% level of confdence.

Detection of the potential TRITC target-protein MIF with immunostaining. Te approximate co-localization of MIF with the TRITC-fuorescent protein band in gel-separated proteins derived from LN cells was confrmed with western blot (see Supplementary Materials online for details). Detection of TRITC-modifed and non-modifed MIF-peptide (PMFIVNTNVPR) in LN fractions using UPLC-qToF. To identify TRITC-bound MIF and non-modifed MIF in the LN single cell suspensions from TRITC-treated mice, the 10–15 kDa section from the gel electrophoresis separation of the proteins was

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 8 www.nature.com/scientificreports/

divided into three pieces (Fig. 3): A – the piece just below the fuorescent band, B – the fuorescent band and C – the piece just above the fuorescent band. In-gel digestion, including reduction with DTT and alkylation with IAA, was performed using Trypsin/Lys-C mix (Promega) according to the manufacturer’s instructions. Te extracted peptides were subsequently concentrated to 30% of the volume using a stream of N2. UPLC-qToF analysis was performed using an Aquity UPLC system (Waters Corporation) coupled to a SYNAPT G2 (Waters MS Technologies) orthogonal acceleration qToF mass spectrometer. The UPLC was equipped with an Aquity CSH C18 column (150 × 2.1 mm i.d., particle size 1.7 μm, Waters Corporation, Milford, MA). Mobile phase A consisted of 0.1% formic acid in water and mobile phase B of 0.1% formic acid in acetonitrile. Aliquots of 10 μl of sample or reference was injected onto the column and eluted with a fow rate of 0.1 ml/ min and a column temperature of 40 °C. Te gradient conditions used were: 0 min 5% B, 10 min 60% B, 11 min 95% B, 12 min 95% B. Te system was equilibrated with 5% B for 3 min between each run. Te qToF was operated in positive resolution mode with electrospray ionization. Te nebulizer gas was set to 800 l/h at a temperature of 450 °C. Te cone gas was set to 100 l/h and the source temperature to 120 °C. Te capillary voltage was set to 3000 V and the sample cone voltage was set to 80 V. A ToF ms/ms method was used where the fxed mass was set to 865.9 or 644.3 m/z, the ms2 was set to 50–1500 m/z and the collision energy ramp was from 15 eV to 40 eV. To ensure mass accuracy, all analyses were acquired using LockSpray with Leucine-enkephaline (556.2771 m/z) as lock mass. Te obtained chromatograms and MS spectra where processed with the smooth function in the MassLynx sofware (Waters Corporation). Te window size was set to ±3 scans and the number of smooths to 5. Te Savitzky Golay method was used for chromatograms and the mean method for MS spectra. To confrm the identity of the non-modifed and TRITC-modifed peptides, synthetic reference compounds were used. A reference mixture was prepared by mixing 200 µl of PMFIVNTNVPR (0.5 mM in sodium phosphate bufer (100 mM) pH 7.5/methanol 1:1) with 50 µl of TRITC (3 mM in methanol) and 750 µl of sodium phosphate bufer pH 7.5, resulting in fnal concentrations of 0.1 mM and 0.15 mM for PMFIVNTNVPR and TRITC, respectively. Te mixture was lef at room temperature for 24 h followed by 10,000-fold dilution with ammonium bicarbonate (25 mM) pH 8.0. With the use of a standard curve of PMFIVNTNVPR the concentrations of PMFIVNTNVPR and TRITC- PMFIVNTNVPR in the reference sample and the gel pieces were estimated (see Supplementary Materials online for details).

Data availability statement. Te datasets generated and/or analysed are available on reasonable request. References 1. Tyssen, J. P., Linneberg, A., Menne, T. & Johansen, J. D. Te epidemiology of contact allergy in the general population–prevalence and main fndings. Contact Dermatitis 57, 287–299, https://doi.org/10.1111/j.1600-0536.2007.01220.x (2007). 2. Peiser, M. et al. Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. Current knowledge assembled at an international workshop at BfR, Germany. Cell Mol Life Sci 69, 763–781, https://doi.org/10.1007/s00018- 011-0846-8 (2012). 3. Martin, S. F. Immunological mechanisms in allergic contact dermatitis. Curr Opin Allergy Clin Immunol 15, 124–130, https://doi. org/10.1097/ACI.0000000000000142 (2015). 4. Kaplan, D. H., Igyarto, B. Z. & Gaspari, A. A. Early immune events in the induction of allergic contact dermatitis. Nat Rev Immunol 12, 114–124, https://doi.org/10.1038/nri3150 (2012). 5. Martin, S. F. New concepts in cutaneous allergy. Contact Dermatitis 72, 2–10, https://doi.org/10.1111/cod.12311 (2015). 6. Landsteiner, K. & Jacobs, J. Studies on the Sensitization of Animals with Simple Chemical Compounds. Ii. J Exp Med 64, 625–639 (1936). 7. Karlsson, I. et al. Peptide Reactivity of Isothiocyanates–Implications for Skin Allergy. Sci Rep 6, 21203, https://doi.org/10.1038/ srep21203 (2016). 8. Basketter, D. A. et al. Local lymph node assay - validation, conduct and use in practice. Food Chem Toxicol 40, 593–598 (2002). 9. Kimber, I. & Weisenberger, C. A murine local lymph node assay for the identifcation of contact allergens. Assay development and results of an initial validation study. Arch Toxicol 63, 274–282 (1989). 10. Gerberick, G. F., Ryan, C. A., Dearman, R. J. & Kimber, I. Local lymph node assay (LLNA) for detection of sensitization capacity of chemicals. Methods 41, 54–60, https://doi.org/10.1016/j.ymeth.2006.07.006 (2007). 11. Dearman, R. J. & Kimber, I. Role of CD4(+) T helper 2-type cells in cutaneous infammatory responses induced by fuorescein isothiocyanate. Immunology 101, 442–451 (2000). 12. Samuelsson, K. et al. Accumulation of FITC near stratum corneum-visualizing epidermal distribution of a strong sensitizer using two-photon microscopy. Contact Dermatitis 61, 91–100 (2009). 13. Kissenpfennig, A. et al. Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells. Immunity 22, 643–654 (2005). 14. Kamath, A. T., Henri, S., Battye, F., Tough, D. F. & Shortman, K. Developmental kinetics and lifespan of dendritic cells in mouse lymphoid organs. Blood 100, 1734–1741 (2002). 15. Kinnaird, A., Peters, S. W., Foster, J. R. & Kimber, I. Dendritic cell accumulation in draining lymph nodes during the induction phase of contact allergy in mice. Int Arch Allergy Appl Immunol 89, 202–210 (1989). 16. Cumberbatch, M. & Kimber, I. Phenotypic characteristics of antigen-bearing cells in the draining lymph nodes of contact sensitized mice. Immunology 71, 404–410 (1990). 17. Simonsson, C., Stenfeldt, A. L., Karlberg, A. T., Ericson, M. B. & Jonsson, C. A. Te pilosebaceous unit–a phthalate-induced pathway to skin sensitization. Toxicol Appl Pharmacol 264, 114–120, https://doi.org/10.1016/j.taap.2012.07.023 (2012). 18. Pior, J., Vogl, T., Sorg, C. & MacHer, E. Free hapten molecules are dispersed by way of the bloodstream during contact sensitization to fuorescein isothiocyanate. J Invest Dermatol 113, 888–893, https://doi.org/10.1046/j.1523-1747.1999.00770.x (1999). 19. Sixt, M. et al. Te conduit system transports soluble antigens from the aferent lymph to resident dendritic cells in the T cell area of the lymph node. Immunity 22, 19–29, https://doi.org/10.1016/j.immuni.2004.11.013 (2005). 20. Su, H., Na, N., Zhang, X. & Zhao, Y. Te biological function and signifcance of CD74 in immune diseases. Infamm Res 66, 209–216, https://doi.org/10.1007/s00011-016-0995-1 (2017). 21. Shimizu, T. Role of macrophage migration inhibitory factor (MIF) in the skin. J Dermatol Sci 37, 65–73, https://doi.org/10.1016/j. jdermsci.2004.08.007 (2005). 22. Calandra, T. & Roger, T. Macrophage migration inhibitory factor: a regulator of innate immunity. Nat Rev Immunol 3, 791–800, https://doi.org/10.1038/nri1200 (2003).

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 9 www.nature.com/scientificreports/

23. Grieb, G., Kim, B. S., Simons, D., Bernhagen, J. & Pallua, N. MIF and CD74 - suitability as clinical biomarkers. Mini Rev Med Chem 14, 1125–1131 (2014). 24. Shimizu, T., Abe, R., Ohkawara, A., Mizue, Y. & Nishihira, J. Macrophage migration inhibitory factor is an essential immunoregulatory cytokine in atopic dermatitis. Biochem Biophys Res Commun 240, 173–178, https://doi.org/10.1006/ bbrc.1997.7633 (1997). 25. Swope, M., Sun, H. W., Blake, P. R. & Lolis, E. Direct link between cytokine activity and a catalytic site for macrophage migration inhibitory factor. EMBO J 17, 3534–3541, https://doi.org/10.1093/emboj/17.13.3534 (1998). 26. Brown, K. K. et al. Direct modification of the proinflammatory cytokine macrophage migration inhibitory factor by dietary isothiocyanates. J Biol Chem 284, 32425–32433, https://doi.org/10.1074/jbc.M109.047092 (2009). 27. Ouertatani-Sakouhi, H. et al. A new class of isothiocyanate-based irreversible inhibitors of macrophage migration inhibitory factor. Biochemistry 48, 9858–9870, https://doi.org/10.1021/bi900957e (2009). 28. Spencer, E. S. et al. Multiple binding modes of isothiocyanates that inhibit macrophage migration inhibitory factor. Eur J Med Chem 93, 501–510, https://doi.org/10.1016/j.ejmech.2015.02.012 (2015). 29. Koppes, S. A. et al. Current knowledge on biomarkers for contact sensitization and allergic contact dermatitis. Contact Dermatitis 77, 1–16, https://doi.org/10.1111/cod.12789 (2017). 30. Simonsson, C. et al. Caged fuorescent haptens reveal the generation of cryptic epitopes in allergic contact dermatitis. J Invest Dermatol 131, 1486–1493, https://doi.org/10.1038/jid.2010.422 (2011). 31. Elbayed, K. et al. HR-MAS NMR spectroscopy of reconstructed human epidermis: potential for the in situ investigation of the chemical interactions between skin allergens and nucleophilic amino acids. Chem Res Toxicol 26, 136–145, https://doi.org/10.1021/ tx300428u (2013). 32. Aleksic, M. et al. Investigating protein haptenation mechanisms of skin sensitisers using human serum albumin as a model protein. Toxicol In Vitro 21, 723–733, https://doi.org/10.1016/j.tiv.2007.01.008 (2007). 33. Parkinson, E. et al. Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers. Toxicol Sci 142, 239–249, https://doi.org/10.1093/toxsci/kfu168 (2014). 34. Dietz, L. et al. Tracking human contact allergens: from mass spectrometric identifcation of peptide-bound reactive small chemicals to chemical-specifc naive human T-cell priming. Toxicol Sci 117, 336–347, https://doi.org/10.1093/toxsci/kfq209 (2010). 35. Jenkinson, C. et al. Characterization of p-phenylenediamine-albumin binding sites and T-cell responses to hapten-modifed protein. J Invest Dermatol 130, 732–742, https://doi.org/10.1038/jid.2009.271 (2010). 36. Rosengren, E. et al. Te immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction. Mol Med 2, 143–149 (1996). 37. Sun, H. W., Bernhagen, J., Bucala, R. & Lolis, E. Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor. Proc Natl Acad Sci USA 93, 5191–5196 (1996). 38. Lubetsky, J. B., Swope, M., Dealwis, C., Blake, P. & Lolis, E. Pro-1 of macrophage migration inhibitory factor functions as a catalytic base in the phenylpyruvate tautomerase activity. Biochemistry 38, 7346–7354, https://doi.org/10.1021/bi990306m (1999). 39. Fleischel, O., Gimenez-Arnau, E. & Lepoittevin, J. P. Nuclear magnetic resonance studies on covalent modifcation of amino acids thiol and amino residues by monofunctional aryl 13C-isocyanates, models of skin and respiratory sensitizers: transformation of thiocarbamates into urea adducts. Chem Res Toxicol 22, 1106–1115, https://doi.org/10.1021/tx9000539 (2009). 40. Nakamura, T., Kawai, Y., Kitamoto, N., Osawa, T. & Kato, Y. Covalent modifcation of lysine residues by allyl isothiocyanate in physiological conditions: plausible transformation of isothiocyanate from thiol to amine. Chem Res Toxicol 22, 536–542, https://doi. org/10.1021/tx8003906 (2009). 41. Ahlfors, S. R., Kristiansson, M. H., Lindh, C. H., Jonsson, B. A. & Hansson, C. Adducts between nucleophilic amino acids and hexahydrophthalic anhydride, a structure inducing both types I and IV allergy. Biomarkers 10, 321–335, https://doi. org/10.1080/13547500500283488 (2005). 42. Nilsson, A. M., Bergstrom, M. A., Luthman, K., Nilsson, J. L. & Karlberg, A. T. A conjugated diene identifed as a prohapten: contact allergenic activity and chemical reactivity of proposed epoxide metabolites. Chem Res Toxicol 18, 308–316, https://doi.org/10.1021/ tx049758c (2005). 43. Niklasson, I. B., Broo, K., Jonsson, C., Luthman, K. & Karlberg, A. T. Reduced sensitizing capacity of epoxy resin systems: a structure-activity relationship study. Chem Res Toxicol 22, 1787–1794, https://doi.org/10.1021/tx900193s (2009). 44. Niklasson, I. B., Delaine, T., Luthman, K. & Karlberg, A. T. Impact of a heteroatom in a structure-activity relationship study on analogues of phenyl glycidyl ether (PGE) from epoxy resin systems. Chem Res Toxicol 24, 542–548, https://doi.org/10.1021/ tx100417r (2011). 45. Bergstrom, M. A., Luthman, K. & Karlberg, A. T. Metabolic epoxidation of an alpha,beta-unsaturated oxime generates sensitizers of extreme potency. Are nitroso intermediates responsible? Chem Res Toxicol 20, 927–936, https://doi.org/10.1021/tx7000114 (2007). 46. Shimizu, T. et al. Impaired contact hypersensitivity in macrophage migration inhibitory factor-defcient mice. Eur J Immunol 33, 1478–1487, https://doi.org/10.1002/eji.200323751 (2003). 47. Pichler, W. J. Direct T-cell stimulations by drugs–bypassing the innate immune system. Toxicology 209, 95–100, https://doi. org/10.1016/j.tox.2004.12.014 (2005). 48. Martin, S. F. et al. T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays. Cell Mol Life Sci 67, 4171–4184, https://doi.org/10.1007/s00018-010-0495-3 (2010). 49. Pichler, W. J. Delayed drug hypersensitivity reactions. Ann Intern Med 139, 683–693 (2003). 50. Yun, J., Cai, F., Lee, F. J. & Pichler, W. J. T-cell-mediated drug hypersensitivity: immune mechanisms and their clinical relevance. Asia Pac Allergy 6, 77–89, https://doi.org/10.5415/apallergy.2016.6.2.77 (2016). 51. Sieben, S., Kawakubo, Y., Al Masaoudi, T., Merk, H. F. & Blomeke, B. Delayed-type hypersensitivity reaction to paraphenylenediamine is mediated by 2 diferent pathways of antigen recognition by specifc alphabeta human T-cell clones. J Allergy Clin Immunol 109, 1005–1011 (2002). 52. Pichler, W. J. Te p-i Concept: Pharmacological Interaction of Drugs With Immune Receptors. World Allergy Organ J 1, 96–102, https://doi.org/10.1097/WOX.0b013e3181778282 (2008). 53. Diamond, D. J. et al. Major histocompatibility complex independent T cell receptor-antigen interaction: functional analysis using fuorescein derivatives. J Exp Med 174, 229–241 (1991). 54. Pichler, W. J., Daubner, B. & Kawabata, T. Drug hypersensitivity: flare-up reactions, cross-reactivity and multiple drug hypersensitivity. J Dermatol 38, 216–221, https://doi.org/10.1111/j.1346-8138.2010.01142.x (2011). 55. Rajasekaran, D. et al. Macrophage Migration Inhibitory Factor-CXCR4 Receptor Interactions: Evidence for Partial Allosteric Agonism in Comparison with CXCL12 Chemokine. J Biol Chem 291, 15881–15895, https://doi.org/10.1074/jbc.M116.717751 (2016). 56. Morand, E. F. et al. Macrophage migration inhibitory factor in rheumatoid arthritis: clinical correlations. Rheumatology (Oxford) 41, 558–562 (2002). 57. de Jong, Y. P. et al. Development of chronic colitis is dependent on the cytokine MIF. Nat Immunol 2, 1061–1066, https://doi. org/10.1038/ni720 (2001). 58. Bernhagen, J. et al. MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature 365, 756–759, https://doi. org/10.1038/365756a0 (1993).

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 10 www.nature.com/scientificreports/

59. Emonts, M. et al. Association between high levels of blood macrophage migration inhibitory factor, inappropriate adrenal response and early death in patients with severe sepsis. Clin Infect Dis 44, 1321–1328, https://doi.org/10.1086/514344 (2007). 60. Mitchell, R. A. Mechanisms and efectors of MIF-dependent promotion of tumourigenesis. Cell Signal 16, 13–19 (2004). 61. Bucala, R. & Donnelly, S. C. Macrophage migration inhibitory factor: a probable link between infammation and cancer. Immunity 26, 281–285, https://doi.org/10.1016/j.immuni.2007.03.005 (2007). 62. O’Reilly, C., Doroudian, M., Mawhinney, L. & Donnelly, S. C. Targeting MIF in Cancer: Therapeutic Strategies, Current Developments and Future Opportunities. Med Res Rev 36, 440–460, https://doi.org/10.1002/med.21385 (2016). 63. Brocks, T. et al. Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin. FASEB J 31, 526–543, https://doi.org/10.1096/f.201600860R (2017). 64. Heise, R. et al. Expression and function of macrophage migration inhibitory factor in the pathogenesis of UV-induced cutaneous nonmelanoma skin cancer. Photochem Photobiol 88, 1157–1164, https://doi.org/10.1111/j.1751-1097.2012.01108.x (2012). Acknowledgements Tis work was fnancially supported by the Swedish Research Council (dnr 2012–4187). We thank Professor Hans Merk from the Department of Dermatology and Allergology, University Hospital RWTH Aachen, Aachen, Germany for valuable suggestions and discussions. Further, we acknowledge Anders Eliasson and Susanne Exing at the Department of Experimental Biomedicine; the Proteomics Core Facility, Sahlgrenska Academy; Maria Smedh and Julia Fernandez-Rodriguez at the Centre for Cellular Imaging; Lillemor Mattsson Hultén and Christina Ullström at the Wallenberg Laboratory for support and use of the BD FACSArray, all from the University of Gothenburg, Gothenburg, Sweden. Author Contributions I.K., K.S., L.L.I., C.J. and A.-T.K. designed the study. I.K., K.S., C.S., C.J. and A.-L.S. performed the experiments and analyzed the data. A.-T.K., L.L.I. and U.N. directed the research. I.K. drafed the manuscript. All authors discussed the results, contributed in manuscript editing and reviewed the fnal manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-21327-8. Competing Interests: Te authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

SCIEntIfIC ReporTS | (2018)8:2895 | DOI:10.1038/s41598-018-21327-8 11